A major difficulty of targeted drug delivery against cancer is the lack of ubiquitously expressed tumour-specific antigen or receptor. However, it has now been established that the phospholipid, Phosphatidylserine (PS) can be exploited as a potential target for drug delivery against cancer.
PS is a phospholipid that under normal conditions resides almost exclusively in the inner leaflet of the plasma membrane. PS asymmetry is maintained by an ATP-dependent amino phospholipid translocase that is responsible for inward movement of the aminophospholipids. Loss of the PS asymmetry is observed under different pathologic and physiologic conditions, including programmed cell death, cell aging, cell migration, cell degranulation (1). Spontaneous PS exposure has been observed in many malignant cell types in the absence of exogenous activators or cell injury (2). Surface PS exposure is observed in solid tumors and tumor vasculature and can be a marker of tumor vasculature (3). PS-expressing tumor endothelium was mostly found to be non-apoptotic. The factors like hypoxic reoxygenation, inflammatory cytokines, acidity, all mostly prevalent factors in tumorigenesis, are mainly the cause of PS exposure. Recent studies have shown that tumorigenic, undifferentiated murine erythroleukemia cell expresses 7-8 fold more PS in their outer leaflet than their differentiated counterparts (2). Elevated expression of surface PS is also found in human melanoma and lung carcinoma (4). Hence, PS on tumor vessels is an attractive target for cancer imaging and therapy. Reports have shown that anti-cancer drugs have been prepared exploiting the elevated PS level of the cancer cells which serves as a marker. NK-lysin derived peptide NK-2 preferentially kills cancer cells. The selectivity of the cationic membranolytic peptide NK-2 has been assigned to the differences in the membrane phospholipid composition of target cancer cells (5). PS exposure is neither of apoptotic nor of experimental artificial origin (6). Anti-phosphatidylserine antibodies have also been used in cancer therapy (7). Hence, it is suggested that the SA-bearing liposomes may prove to be effective in anti-cancer therapy. Since the drug-free liposome itself selectively recognizes and destroys elevated PS exposed on membrane surfaces, this property of the liposome can be utilized in anticancer therapeutic strategies. The SA-bearing liposome has been shown to cause immunomodulation in the host and targets PS-bearing parasites for destruction (8). It is evident that the SA-bearing liposomes should effectively target the cancer cells. Moreover, the efficacy of the anticancer drugs which target the surface PS of the tumor cells and also otherwise shall profoundly increase when administered entrapped in these liposomal formulations. The anticancer drugs frequently affect the normal cells and thus cause severe side effects. But, when administered in a liposomal covering shall thus decrease the cytotoxicity. We can also ensure effective and selective targeting of the anti-cancer drugs when administered within this SA-bearing liposome because PS on the tumor vessels is abundant and is on the luminal surface of the tumor endothelium. This renders it directly accessible for binding by any vasculature targeting agents in the blood. Moreover, it is present on a high percentage of tumor endothelial cells in diverse solid tumors; and it is absent from endothelium in all normal tissues examined to date, thus ensuring selectivity. The phenomenon of PS exposure is not exclusively associated with apoptosis. For instance monocytes differentiating into macrophages and a subpopulation of T-lymphocytes expose PS. Living tumour cells and endothelial cells of the tumour blood vessels also express high levels of PS on their surface. Furthermore cell surface exposure of PS is independent of cell type and thus independent of the type of cancer (3). Vascular-targeted strategies directed against exposed PS may be a powerful adjunct to postoperative chemotherapy in preventing relapses after cancer surgery (9). It has also been shown that B16F10 murine melanoma cells express high levels of PS on their surfaces (10). These features support the proposal that PS can be utilized as an attractive target for the tumour blood vessels as well as the living tumour cells. Liposomes are under extensive investigation as targeted drug delivery against many diseases because the drug here is protected from bioenvironment and thus remains stable for a much longer time. They are small concentric bilayered vesicles, in which an aqueous volume is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids. The first generation of liposomes, a.k.a. conventional liposomes, was developed in the early 70's. They were composed of phosphatidylcholine, phosphatidylserine, phosphoglycerol, and cardiolipine associated with cholesterol so as to reduce permeability (11).
Liposomal Drug Delivery System:
Lipid associated formulations have been proved to be more effective therapies with much lesser toxicity against in vitro and in vivo anti-protozoan activity e.g. visceral leishmaniasis (VL). The advantage of such formulations is their ability to concentrate high levels of drugs in the infected target organs. For example, AmBisome, a liposomal formulation of amphotericin B, is the safest and can be administered at doses much higher than the free form of the drug with much less toxicity (12). The drug-free stearylamine (SA)-bearing cationic liposomes have in vitro activity. A single dose of the drug entrapped in this cationic liposome formulations has a synergistic activity and hence a much more profound effect on the target. The profound effect of the drug-free liposome is due to recognition of the phosphatidylserine (PS) on the parasite membrane by the SA liposomes. The study of the mode of action of the SA-bearing cationic liposomes revealed that the recognition of the surface PS by the liposome is necessary for its ability to damage the parasite membrane resulting in its ultimate death (12). This peculiar mode of action of the drug-free liposomes leads to an interesting hypothesis. SA:PC:Chol in its molar ratio 1:4:5 was found to be toxic for cancer-derived and normal human cultured cell lines at varying degrees (11). Cationic liposomes are used as a delivery system to cells of compounds capable of silencing a target protein and enzyme substrate and also used for detecting inhibitory activity of a target protein in a cell as well as signal transduction pathway in a cell (15).
Classification of Liposomes:
The properties of liposomes vary substantially with lipid composition, size, surface charge and method of preparation. Liposomes can be either classified following in the following manner:
According to Size or Lamellarity
Small Unilamellar Vesicles (<100 nm, a.k.a. SUV) are surrounded by a single lipid bilayer of 25-100 nm diameter. Large Unilamellar Vesicles (100-500 nm, a.k.a. LUV) are a heterogeneous group of vesicles similar to SUVs and are surrounded by a single lipid bilayer. Finally, Multilamellar Vesicles (>500 nm, a.k.a. MLV) consist of several lipid bilayers separated from each other by a layer of aqueous solution. They have onion like structures (11).
According to on the Method of Preparation
Reverse Phase Evaporation Vesicle (REV):
The reverse-phase evaporation technique, the first to use ‘water-in-oil’ emulsions, encapsulates up to 50% of solute. Preparation of reverse-phase evaporation vesicles (REV) consists of a rapid injection of aqueous solution into an organic solvent which contains the lipids dissolved. Thus, following the formation of water droplets (‘water-in-oil’ emulsion) by bath sonication of the two-phase mixture, the emulsion is dried down to a semi-solid gel in a rotary evaporator. The next step is to subject the gel to vigorous mechanical shaking to induce a phase change from a water-in-oil emulsion to a vesicle suspension. In these circumstances, some water droplets collapse, and these droplets attach to adjacent, intact vesicles to form the outer leaflet of the bilayer of a large unilamellar liposome (diameter in the range of 0.1 to 1 μm) (16).
Dehydration-Rehydration Vesicles (DRV):
Another method that produces dehydration-rehydration vesicles (DRV) is both simple and easy to scale up, and usually gives high yields of solute entrapment (up to 80%). Preparation of DRV consists of mixing an aqueous solution of the solute with a suspension of ‘empty’ (water-containing) liposomes and freeze-drying the resulting, mixture. The intimate contact of flattened liposomal membrane structures and solute molecules in a dry environment and the fusion of membranes caused by dehydration facilitates the incorporation of solute during the controlled rehydration steps. Separation of solute-containing DRV from unentrapped solute can be carried out by centrifugation easily if needed. Vesicles formed by the dehydration-rehydration technique are multilamellar with heterogeneous sizes (diameters varying from 0.1 to 2.0 μm) (16).
Multilamellar Vesicles (MLV):
The most easily prepared and processed liposomal form is Multilamellar vesicles or MLVs. MLVs are prepared by first casting a lipid film in organic solvent (chloroform). Then lipid particles are dispersed in aqueous solvent followed by probe sonication. This form of liposome has advantages over both DRV and REV form of liposome due to its optimum size (200-250 nm) and multilamellar structure which allows higher amount of drug to be successfully entrapped into the liposome. The MLV form of liposome showed maximum efficacy in the present model and highest drug entrapment efficacy. Hence, most of the experiments were performed with this particular form of liposome.
According to In Vivo Application:
Conventional liposomes which may be neutral or negatively charged. They are used mainly for macrophage targeting or for vaccination. Stealth (stearically stabilized) liposomes which carry polymer coatings to obtain prolonged circulation times. Immunoliposomes (antibody targeted) are used for specific targeting and Cationic liposomes are employed mainly for gene delivery or cancer therapy. Liposomes containing cationic lipids (DOTAP:DOPE, SA) have been widely used as transfection mediators both in vitro and in vivo due to their ability to interact with negatively charged molecules such as DNA and phosphatidylserine of cell membranes. The amphiphilic properties of cationic lipid molecules together with positive charge and defined phase behaviour of liposomes composed of them, make possible interactions such as adsorption, fusion, poration and destabilization with negatively charged membranes. Some cationic lipids are able to penetrate natural membranes and localize in the inner leaflet, forming invaginations and even endosome-like vesicles. The initial event occurring between cationic liposomes and negatively charged plasma membrane is adsorption. Electrostatic and hydrophobic interactions may lead to hemifusion, fusion, poration or, alternatively, receptor-mediated endocytosis may occur (17).
Biomembrane mimetic model system were prepared with PC:PS, PC:PA or PC:Chol liposomes and it was found that PC:SA liposomes had specific affinity for PC:PS liposomes rather than PC:Chol or anionic PC:PA liposomes. This further supported the PC:SA liposome interacted mainly with PS. This indicates that the SA-bearing liposomes can be used as a valuable delivery system against cancer cells which also have elevated levels of negatively charged PS on the outer surface of their membranes (12). Even in cationic liposomes variations can be made on the basis of methods of preparation. Previously, work has been done on comparative study of these different types of liposomes and their antigen entrapment efficiency with Leishimania donovani promastigote membrane antigens (LAg). In that study mice were immunized with LAg encapsulated in multilamellar vesicles (MLV), dehydration-rehydration vesicles (DRV) and reverse-phase evaporation vesicles (REV) and were challenged with parasites ten days after vaccination. Leishmanial antigen (LAg) in MLV or DRV induced almost complete protection, while LAg alone or entrapped in REV exhibited partial resistance. MLV encapsulated LAg demonstrated durable cell-mediated immunity and mice challenged ten weeks after vaccination could also resist experimental challenge strongly (18).
Selecting an Appropriate Antineoplastic Agent:
Most patients with advanced solid tumours still die of their disease. For this reason, new effective drugs are needed. A number of anticancer drugs are under investigation at present which target the tumor cell at DNA or protein level. Other elements interacting with tumors like the endothelium or extracellular matrix may also be targeted.
Camptothecin (CPT) is a quinoline alkaloid isolated from the bark and stem of Camptotheca acuminata (Camptotheca, Happy tree), a tree native to China (19). It exhibits potent cytotoxic activity against a range of tumor cell lines CPT, possesses a high melting point (264-267° C.), and has a molecular weight of 348.11 obtained by high-resolution mass spectroscopy, corresponding to the formula (C20H16N2O4). Camptothecin inhibits both DNA and RNA synthesis in mammalian cells. The inhibition of RNA synthesis results in shortened RNA chains and is rapidly reversible upon drug removal while inhibition of DNA synthesis is only partially reversible. CPT binds to Topoisomerase 1 and DNA complex (with Hydrogen bonds), resulting in a stable ternary complex. Topoisomerases are a family of enzymes which relax supercoiled DNA by making transient single stranded breaks in the DNA, allowing the uncut strand to pass through the break before resealing the nick (thus increasing its linking number by 1). Binding of CPT inhibits this rejoining step which ultimately leads to DNA fragmentation and apoptosis. This drug is widely distributed in the body including the central nervous system, lungs and liver (20).
Camptothecin encloses in its structure a highly conjugated pentacyclic ring with an α-hydroxylactone portion at carbon 12 which is essential for its in vitro and in vivo antitumor activity. Unfortunately this lactone ring is highly susceptible to hydrolysis and under physiological conditions i.e., at pH 7 or above, the lactone ring readily opens to yield the inactive carboxylate form of the drug (20). Ring opening of camptothecin is thought to result in a loss of activity due to the following three reasons. First, the carboxylate form displays decreased association with the membrane. Second, ring opening results in a charged drug species which exhibits limited diffusibility through lipid bilayer domains. Third, evidence from cell-free experiments indicates that ring opening results in significantly reduced intrinsic potency towards the topoisomerase-1 target.
The above drawback along with poor water solubility and high adverse drug reaction limits the application of CPT in therapeutics. For this reason different lipid based formulations were investigated and it was found that CPT is soluble in various lipids and also biologically active at the same time. Two types of spectroscopic data are available which support that liposome associated camptothecin is stable. The first evidence comes from, where there is a blue shift in the drug's emission spectrum which is observed upon its association with membrane. Such a spectral shift is indicative of a change in the dielectric constant of the medium surrounding the fluorophore, as when a compound leaves an aqueous environment and intercalates in between the lipid acyl chains (21). Thus, liposomal drug delivery systems are of potential utility for introducing camptothecin (or related lipophillic analogues) in its stable and pharmacologically active form to cancer victims.
Doxorubicin (DOX) is another an important class of drug that is used in cancer therapy. It is an anthracycline antibiotic. It is photosensitive in nature. It was first extracted from Streptomyces peucetius. It is used in the treatment of several cancers that include breast cancer, lung, ovarian, gastric, Hodgkin's and Non Hodgkin's lymphoma, Multiple myeloma and sarcoma and pediatric cancers. The main function of doxorubicin is in intercalating DNA. There are two proposed mechanisms by which doxorubicin acts on cancer cells. They are (i) Intercalation into DNA and disruption of topoisomerase II mediated DNA repair and (ii) Generation of free radicals and their disruption of cell membrane and damage to DNA and proteins. In other words doxorubicin is oxidized to semiquinone (an unstable metabolite) which is converted back to doxorubicin in a process that releases reactive oxygen species (ROS). ROS have the ability to cause lipid peroxidation and membrane damage, DNA damage, oxidative stress, and stimulates apoptic pathways of cell death. Doxorubicin can enter the nucleus, poison DNA topoisomerase II and cause damage to the DNA and cell death. A major limitation for the use of doxorubicin is cardiotoxicity, with the total cumulative dose being the only criteria currently used to predict the toxicity (22).
The present invention claims that cationic liposome in all its three forms (MLV, DRV, REV) is a potential anticancer agent which selectively targets the cancer cells through the cancer cell surface exposed phosphatidylserine. The formulation has no adverse effects on the normal cells and hence is a successful targeted therapy against cancer. The anticancer drug encapsulated formulations had significant anticancer effect both in vitro and in vivo. The drug encapsulated liposome has a synergistic effect and hence shall be effective in bringing down the dosage of the drug and thus protecting against chemotherapeutic toxicity. The liposomal formulation is successful in controlling the adverse chemotherapeutic effect of anti-cancer drugs due low dosage of application with high efficacy and targeted delivery with minimum damage to normal cells. The formulation mainly works through the intrinsic kinase signalling pathway of the cancer cells. It can be a valuable therapeutic agent since it showed negligible therapeutic toxicity and miraculous therapeutic efficiency in vivo.